Link to this post | posted 24 May, 2019 21:12
scaruso	Ah. Thanks. That helps. Steve Edited 24 May, 2019 21:13

Link to this post | posted 24 May, 2019 18:04

scaruso

I am checking a BE1 submission, which have 40+ tRNAs. I noticed, as I go though them that I would not have accepted all of these. I've already found four with acceptor stems of 5 bp, 6 bp, and 8 bp, and a couple with very low scores (10 and 14), but that are present in other BE1 phages. Before I deleted them, I wanted to make sure there was no reason these were in there outside the guidlines. Thanks! Steve Edited 24 May, 2019 18:08

Link to this post | posted 20 May, 2019 21:13

scaruso

We certainly had problems with smears in the past. Most of the time, I have attributed it to user error, and we have switched to adding EDTA and proteinase K to our protocol. It has nearly, but not completely eliminated the issue. I think the few that remain are a mixture mostly of students that are incautious, and a few that are something else. We have phages that simply refuse to give us pretty DNA (the Bacillus phi29-like podos are a reliable example) unless we use phenol chloroform despite very high titers. That's consistent over several years among many students. We have to wonder if there is something interesting going on with their DNA or capsid, or a packaged enzyme that is being denatured by the phenol but not by the standard isolation. Then, this year, we had several, five I think, Streptomyces phages that were not cut at all when tested by every enzyme I could get my hands on (I think I had ten or so). These were enzymes that were consistently cutting other phages' DNA, so we knew they worked. So, not a smear, but modified DNA? Seems like a possibility. Steve

Link to this post | posted 16 May, 2019 18:56
scaruso	OK, so, after further consideration and looking at the second one, I think calling the first gene Lysin A is probably pretty safe. I don't know about calling the second one anything beyond hydrolase. Steve

Link to this post | posted 13 May, 2019 17:03

scaruso

These phages have a second gene identified as hydrolase immediately following two minor tail proteins and immediately before the holin, so it is hard to tell if it is another minor tail protein or a lysin by synteny, so the more generic call has been used in the past. That is - https://toolkit.tuebingen.mpg.de/#/jobs/Esketit_28a and https://toolkit.tuebingen.mpg.de/#/jobs/Esketit_28b Once this is resolved, it's ready, by the way. Steve Edited 13 May, 2019 17:03

Link to this post | posted 12 May, 2019 01:09

scaruso

I am reviewing a BI1 and have a another call that I am planning on deleting, thymidylate kinase. The call has been made based on a blast hit to several other phages, but I can't find any other evidence that it really is thymidylate kinase. If this were one of our phages, I would call it NKF for sure, but wanted to get a second opinion since it is the first one I am reviewing externally. Three members of pham 44619 (https://phagesdb.org/phams/44619/) have the call, the rest don't. The results for the gene are here https://toolkit.tuebingen.mpg.de/#/jobs/Esketit_73a and https://toolkit.tuebingen.mpg.de/#/jobs/Esketit_73b. No hint of a hit other than by blast - https://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Get&RID=DEW588MA015. I think they are questionable hits. But the approved functions list uses Erdmann_94 as the example of the call, which is pham 46029 - https://phagesdb.org/phams/46029/, and has been changed to thymidylate synthase and is mostly filled with ThyX, etc. Upshot is, I think the kinase call wrong. Thoughts? Steve Edited 13 May, 2019 00:01

Link to this post | posted 11 May, 2019 17:36

scaruso

I am reviewing a BI1 and have a couple genes with functions assigned that are not on the official function list. Both were undoubtedly chosen because they were assigned historically. I just want to get a backstop of my plan. I'll post them as separate posts. One is endolysin, which I am changing to hydrolase. Eleven cluster BI phages have the function endolysin assigned to a gene in pham 3570 (https://phagesdb.org/phams/3570/) The protein does hits a bunch of previous endolysin and hydrolase calls (https://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Get&RID=DE1EXX8D015) and has a lysozyme domain when blasted. HHPred shows a strong lysozyme domain on the right side, and a weaker peptidase/hydrolase domain on the left (https://toolkit.tuebingen.mpg.de/#/jobs/Esketit_5a) and (https://toolkit.tuebingen.mpg.de/#/jobs/Esketit_5b) I see a general call of hydrolase as the safest choice. It has two domains, so I considered Lysin A, but I'm not confident in it. Thoughts? Steve Edited 11 May, 2019 20:19

Link to this post | posted 11 May, 2019 17:20

scaruso

I am reviewing a BI1 and have a couple genes with functions assigned that are not on the official function list. Both were undoubtedly chosen because they were assigned historically. I just want to get a backstop of my plan. I'll post them as separate posts. One is chitosinase, which I am changing to glycoside hydrogenase. Eight cluster BI phages have the function chitosinase assigned to a gene in pham 16099 (https://phagesdb.org/phams/16099/). The protein does hit chitosinase in Strep and other bacterial spp. by both blastp (https://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Get&RID=DE09SEP6015) and HHPred (https://toolkit.tuebingen.mpg.de/#/jobs/Esketit_22a) and (https://toolkit.tuebingen.mpg.de/#/jobs/Esketit_22b). The closest approved function is a glycoside hydrogenase, of which chitosinase is a type. The other option is to put in a petition to have it added to the list. Thoughts? Steve Edited 11 May, 2019 17:40

Link to this post | posted 05 Apr, 2019 13:12

scaruso

Good morning, We have some mass spec data to look at and are being told that it likely is suffering from all sorts of post translational modifications, which make it harder for their system to match up the results to the provided data, it seems. Unless, of course, we tell them which to look for. Oh and each one included adds to the computing time. So best to chose judiciously. OK, so, with that in mind, we have come up with only a couple common one identified in the host bacteria. Strep apparently does a lot of acetylation, adenylylation, and modification with prokaryotic ubiquitin-like protein (Pup). But anyone have some suggestions of the best choices to include for Actinobacteriophages in general? Thanks, Steve

Link to this post | posted 25 Mar, 2019 16:45
scaruso	We used the function "major capsid and protease fusion protein," the current version of "capsid & capsid maturation protease" of the cluster AM phages (see Pham 17557). Should another function be created, perhaps that recognizes all three domains? Steve